Proximity labeling defines the phagosome lumen proteome of murine and primary human macrophages
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Abstract
Proteomic analyses of the phagosome has significantly improved our understanding of the proteins which contribute to critical phagosome functions such as apoptotic cell clearance and microbial killing. However, previous methods of isolating phagosomes for proteomic analysis have relied on cell fractionation with some intrinsic limitations. Here, we present an alternative and modular proximity-labeling based strategy for mass spectrometry proteomic analysis of the phagosome lumen, termed PhagoID. We optimize proximity labeling in the phagosome and apply PhagoID to immortalized murine macrophages as well as primary human macrophages. Analysis of proteins detected by PhagoID in murine macrophages demonstrate that PhagoID corroborates previous proteomic studies, but also nominates novel proteins with unexpected residence at the phagosome for further study. A direct comparison between the proteins detected by PhagoID between mouse and human macrophages further reveals that human macrophage phagosomes have an increased abundance of proteins involved in the oxidative burst and antigen presentation. Our study develops and benchmarks a new approach to measure the protein composition of the phagosome and validates a subset of these findings, ultimately using PhagoID to grant further insight into the core constituent proteins and species differences at the phagosome lumen.
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Because the phagosome preparations from iBMDM and hMDM were prepared identically in this study, we reasoned that we could gain insight into distinct phagosomal features between the macrophages derived from two different species.
Are you confident that dfferences between iBMDM and hMDM PhagoID results are species and not related to differences between primary cells and a cell line?
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Several proteins identified by PhagoID were annotated as nuclear proteins that raise new hypotheses about phagosome identity and function
Have you validated the presence of nuclear-annotated proteins in the phagosome with alternative low-throughput methods?
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We tested if enzymatic removal of N-linked glycans would augment detection of proteins labeled by PhagoID by eliminating the need to search for the many possible glycan structures at each glycosylation site
It is reasonable to assume that different glycosylation of at least some phagosomal proteins carries specific functional importance beyond generic resistant to proteolysis. Could your enrichment method also be used to enable glycoproteomic analysis of phagosome contents?
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we also generated a bead-based cargo of 3 μm beads with HRP chemically conjugated on their surface
Is there a reason you used a bead-based HRP instead of putting it on a more biologically-relevant phagocytic cargo? Because this introduces an additional variable, did you do any controls to show that the superior activity of HRP is not due to the bead?
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Because of its highly dynamic and interactive nature, the phagosome does not exist as an independent organelle, but instead as a mosaic of multiple contributing cell compartments.
Is it possible to use PhagoID technology to study the development and evolution of the phagosome over time?
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